Genetic Construction of Bacillus subtilis Recombinant Strain, Producing Anthrax Protective Antigen

Anthrax is a serious infectious disease with high mortality. The epidemiological security depends on the vaccination of susceptible animals and population at risk. But many of the existing anthrax vaccine strains possess low levels of protective antigen production and high reactogenicity. One of the most promising trends in production of new generation of vaccines is the cloning of particular determinants of immunogenicity of anthrax microbe for the creation of highly effective producers of Bacillus anthracis protective antigen. The aim of the article is to present the results of the study on the construction of recombinant Bacillus subtilis strain, producing B.anthracis protective antigen, promising for use in chemical anthrax vaccines technology. The pHT43PA plasmid containing the gene pag, providing the synthesis of protective antigen of the anthrax microbe and functioning stably in the cells of the recombinant strain Amy21(pHT43РА) of B. subtilis, was constructed on the basis of the shuttle vector pHT43. It is found out during the research, that the microbial cells of the recombinant strain Amy21(pHT43РА) of B. subtilis provide the production of immunologically active protective antigen in quantities, not inferior than anthrax vaccine strains. These data, as well as safety and simplicity of В. subtilis make it possible to continue the research of this recombinant strain as a producer of anthrax protective antigen, promising for use in vaccines production

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Mechanistic Analysis of the Effect of Deamidation on the Immunogenicity of Anthrax Protective Antigen

Clinical and Vaccine Immunology ◽

10.1128/cvi.00701-15 ◽

2016 ◽

Vol 23 (5) ◽

pp. 396-402 ◽

Cited By ~ 12

Author(s):

Anita Verma ◽

Miriam M. Ngundi ◽

Drusilla L. Burns

Keyword(s):

T Cell ◽

Antibody Production ◽

Protective Antigen ◽

Primary Component ◽

Wild Type ◽

Immunological Mechanisms ◽

Anthrax Protective Antigen ◽

T Cell Help ◽

New Generation ◽

Anthrax Vaccines

ABSTRACTThe spontaneous modification of proteins, such as deamidation of asparagine residues, can significantly affect the immunogenicity of protein-based vaccines. Using a “genetically deamidated” form of recombinant protective antigen (rPA), we have previously shown that deamidation can decrease the immunogenicity of rPA, the primary component of new-generation anthrax vaccines. In this study, we investigated the biochemical and immunological mechanisms by which deamidation of rPA might decrease the immunogenicity of the protein. We found that loss of the immunogenicity of rPA vaccine was independent of the presence of adjuvant. We assessed the effect of deamidation on the immunodominant neutralizing B-cell epitopes of rPA and found that these epitopes were not significantly affected by deamidation. In order to assess the effect of deamidation on T-cell help for antibody production elicited by rPA vaccine, we examined the ability of the wild-type and genetically deamidated forms of rPA to serve as hapten carriers. We found that when wild-type and genetically deamidated rPA were modified to similar extents with 2,4-dinitrophenyl hapten (DNP) and then used to immunize mice, higher levels of anti-DNP antibodies were elicited by wild-type DNP-rPA than those elicited by the genetically deamidated DNP-rPA, indicating that wild-type rPA elicits more T-cell help than the genetically deamidated form of the protein. These results suggest that a decrease in the ability of deamidated rPA to elicit T-cell help for antibody production is a possible contributor to its lower immunogenicity.

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Identification of a Protein Subset of the Anthrax Spore Immunome in Humans Immunized with the Anthrax Vaccine Adsorbed Preparation

Infection and Immunity ◽

10.1128/iai.73.9.5685-5696.2005 ◽

2005 ◽

Vol 73 (9) ◽

pp. 5685-5696 ◽

Cited By ~ 28

Author(s):

Indira T. Kudva ◽

Robert W. Griffin ◽

Jeonifer M. Garren ◽

Stephen B. Calderwood ◽

Manohar John

Keyword(s):

Drug Targets ◽

Protective Antigen ◽

Hypothetical Protein ◽

The United States ◽

Surface Proteins ◽

Spore Surface ◽

Expression Library ◽

Anthrax Vaccine ◽

Anthrax Spore ◽

Anthrax Vaccines

ABSTRACTWe identified spore targets of Anthrax Vaccine Adsorbed (AVA)-induced immunity in humans by screening recombinant clones of a previously generated, limited genomicBacillus anthracisSterne (pXO1+, pXO2−) expression library of putative spore surface (spore-associated [SA]) proteins with pooled sera from human adults immunized with AVA (immune sera), the anthrax vaccine currently approved for use by humans in the United States. We identified 69 clones that reacted specifically with pooled immune sera but not with pooled sera obtained from the same individuals prior to immunization. Positive clones expressed proteins previously identified as localized on the anthrax spore surface, proteins highly expressed during spore germination, orthologs of proteins of diverse pathogens under investigation as drug targets, and orthologs of proteins contributing to the virulence of both gram-positive and gram-negative pathogens. Among the reactive clones identified by this immunological screen was one expressing a 15.2-kDa hypothetical protein encoded by a gene with no significant homology to sequences contained in databases. Further studies are required to define the subset of SA proteins identified in this study that contribute to the virulence of this pathogen. We hypothesize that optimal delivery of a subset of SA proteins identified by such studies to the immune system in combination with protective antigen (PA), the principal immunogen in AVA, might facilitate the development of defined, nonreactogenic, more-efficacious PA-based anthrax vaccines. Future studies might also facilitate the identification of SA proteins with potential to serve as targets for drug design, spore inactivation, or spore detection strategies.

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Biosynthesis of γ-aminobutyric acid by a recombinant Bacillus subtilis strain expressing the glutamate decarboxylase gene derived from Streptococcus salivarius ssp. thermophilus Y2

Process Biochemistry ◽

10.1016/j.procbio.2014.08.007 ◽

2014 ◽

Vol 49 (11) ◽

pp. 1851-1857 ◽

Cited By ~ 6

Author(s):

Chong Zhang ◽

Jing Lu ◽

Lin Chen ◽

Fengxia Lu ◽

Zhaoxin Lu

Keyword(s):

Bacillus Subtilis ◽

Glutamate Decarboxylase ◽

Aminobutyric Acid ◽

Subtilis Strain ◽

Streptococcus Salivarius ◽

Recombinant Bacillus Subtilis

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Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain

Microbiology ◽

10.1134/s0026261706020068 ◽

2006 ◽

Vol 75 (2) ◽

pp. 142-147 ◽

Cited By ~ 1

Author(s):

Yu. M. Kirillova ◽

E. O. Mikhailova ◽

N. P. Balaban ◽

A. M. Mardanova ◽

A. R. Kayumov ◽

...

Keyword(s):

Bacillus Subtilis ◽

Serine Proteinase ◽

Subtilis Strain ◽

Bacillus Intermedius ◽

Recombinant Bacillus Subtilis

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Immunization against anthrax with aromatic compound-dependent (Aro-) mutants of Bacillus anthracis and with recombinant strains of Bacillus subtilis that produce anthrax protective antigen.

Infection and Immunity ◽

10.1128/iai.58.2.303-308.1990 ◽

1990 ◽

Vol 58 (2) ◽

pp. 303-308 ◽

Cited By ~ 95

Author(s):

B E Ivins ◽

S L Welkos ◽

G B Knudson ◽

S F Little

Keyword(s):

Bacillus Subtilis ◽

Bacillus Anthracis ◽

Aromatic Compound ◽

Protective Antigen ◽

Recombinant Strains ◽

Anthrax Protective Antigen

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Surface Display of Antigen Protein VP8* of Porcine Rotavirus on Bacillus Subtilis Spores Using CotB as a Fusion Partner

Molecules ◽

10.3390/molecules24203793 ◽

2019 ◽

Vol 24 (20) ◽

pp. 3793 ◽

Cited By ~ 3

Author(s):

Wanqiang Li ◽

Jie Feng ◽

Jiajun Li ◽

Jianzhen Li ◽

Zhenhua Wang ◽

...

Keyword(s):

Bacillus Subtilis ◽

Recombinant Strain ◽

Shuttle Vector ◽

Surface Display ◽

Mucosal Vaccine ◽

Viral Gastroenteritis ◽

Fusion Partner ◽

Spore Surface ◽

Effective Measure ◽

Porcine Rotavirus

Porcine rotavirus is a major cause of acute viral gastroenteritis in suckling piglets, and vaccination is considered to be an effective measure to control these infections. The development of a live mucosal vaccine using Bacillus subtilis spores as an antigen delivery vehicle is a convenient and attractive vaccination strategy against porcine rotavirus. In this study, a shuttle vector was constructed for the spore surface display of the spike protein VP8* from porcine rotavirus (the genotype was G5P[7]). A successful display of the CotB-VP8* fusion protein on the spore surface was confirmed by Western blot and immunofluorescence microscopy analysis. The capacity for immune response generated after immunization with the recombinant strain was evaluated in a mouse model. The intestinal fecal IgA and serum IgG were detected by enzyme-linked-immunosorbent serologic assay (ELISA). Importantly, recombinant strain spores could elicit strong specific mucosal and humoral immune responses. These encouraging results suggest that recombinant B. subtilis BV could provide a strategy for a potential novel application approach to the development of a new and safe mucosal subunit vaccine against porcine rotavirus.

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